The purpose of these studies is to establish a better understanding of the energy metabolism in tissues, in vivo. Towards this goal, the laboratory concentrates on the use of non-invasive and non-destructive techniques to evaluate the biochemical and physiological function of the heart and skeletal muscle with regard to energy metabolism. The following major findings were made over the last year: 1)It was demonstrated that the sarcoplasmic reticulum (SR) and the mitochondria share similar steady state kinetics for calcium ion (Ca) activation. In a reconsistuted SR and mitochondria preparation, Ca additions were shown to result in a balanced activation of SR ATP hydrolysis and ATP production in the mitochondria resulting in a 5 fold increase in flux without significant changes in the metabolic intermediates. The temporal response of these two systems to Ca was also found to be nearly identical. It is proposed that the balanced kinetics of Ca activiation of the SR and mitochondria is responsible for a maintenance of high energy phosphates in the heart with changes in workload. 2) Previously this laboratory had demonstrated the use of fluorescence photo-bleaching methods to monitor the the dehydrogenase activity within single living cells. This is accomplished by selectively removing a metabolite, NADH, from the cell with an optical pulse and observing its recovery via the dehydrogenase enzyme system. This processes is called enzyme dependent fluorescence recovery after photobleaching (ED-FRAP). Using a newly developed in vitro system, we have discovered that the photobleaching of NADH by the optical pulse causes the simple oxidation of NADH to NAD. Thus the photobleaching process does not destroy the molecule but permits it to be recycled in the cell. This conclusion was supported by NMR, mass spectroscopy and enzymology studies. The other products of the NADH photolysis reaction include protons and free electrons depending on the environmental conditions. Using this information a more complete picture of the photobleaching process has been established for quantitative studies within living cells and organelles 3) In isolated mitochondria NADH ED-FRAP has been shown to be highly dependent on Ca suggesting that the dehydrogenase system is under tight regulatory control by Ca levels in the matrix. This is the first direct demonstration of dehydrogenase activiation by Ca in isolated mitochondria supporting the role of Ca in the overall cellular regulation of energy metabolism in the heart cell. 4)The possible existence of nitric oxide synthetase in heart mitochondria was evaluated to establish whether the local modulation of mitochondrial NO could affect oxidative phosphorylation. Based on both functional and protein identification studies, it was concluded that the nitric oxide synthetic activity as small in porcine heart mitochondria and did not have physiological significance in the regulation of ATP production.